This R21 application focuses on providing critical new insights into the mechanism by which the predominant extracelluar enamel matrix protein, amelogenin, regulates initial enamel mineral formation and tissue organization. Based on extensive prior findings, our overall hypothesis is that phosphorylation of serine-16 (S-16) modulates the structure and hierarchical assembly of native (phosphorylated) full-length amelogenin, resulting in its greatly enhanced capacity to stabilize amorphous calcium phosphate (ACP) nanoparticles. However, this hypothesis is based solely on in vitro findings and the importance of the role of amelogenin phosphorylation in vivo is unknown and essentially unexplored. The goal of this R21 application is to explore this understudied area and to develop necessary tools to test this hypothesis directly in vivo and provide the basis and means for further studies on the specific role of amelogenin phosphorylation in regulating enamel formation. Two (2) multi-dimensional specific aims have been proposed to achieve these goals. Specifically, we propose: in Aim 1. To develop a novel mouse model in which amelogenin phosphorylation does not occur, to be used to assess the functional consequences of amelogenin phosphorylation in enamel development in vivo. A knock-in (KI) mouse model will be developed through the construction of a targeting vector in which a point mutation of the sole S-16 phosphorylation site for alanine is engineered in a constitutive manner; and in Aim 2. To assess the effect of altered amelogenin on the phase, morphology and structural organization of developing enamel mineral in KI mice that generate non-phosphorylated amelogenin, in comparison to that produced in WT littermates. These latter studies will be carried out to test the hypothesis that phosphorylation of the native full-length amelogenin is required to promote the stabilization, proper alignment and transformation of ACP to ordered apatitic crystals, as seen in vivo. Multiple approaches will be used to characterize the KI mouse, including routine histology, PCR followed by digestion with restriction enzymes, RT-PCR, and Western blot analyses. Developing teeth in KI and WT mice will be characterized using TEM, selected area electron diffraction, Raman microspectroscopy, SEM, micro-CT and via micro-hardness measurements. The proposed studies are designed to provide fundamental insight into how matrix proteins, like amelogenin, control mineralization and structure in mineralized tissues. As a long-term goal, our findings should aid in the development of novel approaches for the regeneration and repair of diseased or damaged dental enamel. Given the high prevalence of dental caries, there is a tremendous need for restorative procedures that are superior to those presently available. Findings obtained from studies proposed in this R21 application will serve as a basis for future investigations on the regulation of enamel formation in vivo.